Stimulation of transcription in vitro from a liver-specific promoter by human glucocorticoid receptor (hGRalpha).

The rat tyrosine aminotransferase (TAT) gene is a liver-specific and glucocorticoid-inducible gene. Previous studies have shown that the TAT promoter (TAT0.35; nt -350 to +1) is able to sustain liver-specific gene expression both in transient transfection and in a transcription assay in vitro [Schweizer-Groyer, Groyer, Cadepond, Grange, Baulieu and Pictet (1994) Nucleic Acids Res. 22, 1583-1592]. Here we report that the basal transcriptional activity generated from TAT0.35 in the presence of crude liver nuclear extracts is enhanced by added human glucocorticoid receptor (hGRalpha), provided that TAT0.35 sequences were flanked (5') with a glucocorticoid responsive unit (GREII of the TAT gene, including its 5'-CCAAT flanking sequence). Two sources of hGRalpha were used: nuclear extracts prepared from Sf9 insect (Sf9-NEs) cells over-expressing hGRalpha, and hGRalpha from pRShGRalpha-transfected COS-7 cells, enriched by high-performance ion-exchange chromatography. The enhancement of transcription in vitro (1.5-4.5-fold) was dependent on the amount of added hGRalpha and independent of the nature (agonist or antagonist) of the ligand. Moreover, the hGRalpha-mediated stimulation of transcription was (i) dependent on GRE/progesterone response element (PRE) (it was inhibited by a 25-fold excess of GRE/PRE but not by a 100-fold excess of oestrogen response element) and (ii) receptor-dependent (Sf9-NEs prepared from uninfected Sf9 cells or from Sf9 cells infected with wild-type baculoviral DNA did not enhance transcription). Taken together, these experiments support the conclusions that in vitro the glucocorticoid receptor is able to enhance transcription from genomic, liver-specific, promoter sequences (those of the TAT gene), and that this enhancement of transcription from the liver-specific TAT0.35 promoter is dependent both on the glucocorticoid receptor and on the latter's interaction with its cognate response elements.

Glucocorticosteroid receptor dimerization investigated by analysis of receptor binding to glucocorticosteroid responsive elements using a monomer-dimer equilibrium model.

The aim of this study was to analyze the role of regions of the glucocorticosteroid receptor (GR) outside the DNA binding domain (DBD) in GR binding and homodimerization efficiencies by using a model according to which GR monomers and dimers are in equilibrium and able to bind to each half-palindromic motif of a GRE. We studied wild-type human GR (hGR), an N-terminal domain deleted mutant (lacking amino acids 1-417), a C-terminal deleted mutant (lacking amino acids 550-777, the main part of the ligand binding domain), and two rat GR derivatives limited to the DNA binding domain and proximal sequences. Specific GR monomer and dimer complexes with 33P-labeled palindromic or half-palindromic GREs were identified by gel-shift and methylation interference experiments. The different complexes were quantified, and the multiple equilibrium constants for their formation were determined. The affinity of the monomer for the GRE was not affected by the deletions of regions outside the DBD. However, the affinity of the dimer for the GRE was clearly increased by the presence of the N-terminal domain and, to a lesser extent, by that of the main part of the C-terminal domain. By using this model, we also obtained a GR dimerization constant in the absence of specific binding to GRE. Dimerization of the DBD was not increased by the presence of only one of the GR terminal domains, but an increase in dimerization efficiency was observed when both domains were present, suggesting a structural synergy between the N- and C-terminal domains in GR homodimerization.

Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticosteroid receptor (GR) or hormone binding by GR.

We have developed an in vivo system using coexpression of human glucocorticosteroid receptor (hGR) and chick hsp90 alpha (chsp90) in recombinant virus-infected Sf9 cells to study the formation of hetero-oligomeric complexes. We detected, in the cytosol, hGR complexes containing chsp90 as shown by the displacement of the [3H]triamcinolone acetonide bound hGR "8S" peak on glycerol/sucrose gradients by specific antibodies directed against chsp90 (BF4 and D7 alpha). We took advantage of this system and of the immunoadsorption of hGR containing complexes with anti-hGR antibody to analyze the effect of deletions introduced into the hsp90 molecule on the formation of complexes with the hGR. Deletion of the hydrophilic region "A", between amino-acids 221 and 290, abolished the formation of hGR/chsp90 complexes. Deletion of the hydrophilic region "B" (between amino-acids 530 and 581) or deletion of a leucine repeat region "Z" in the middle of the molecule (amino-acids 392 to 419) still allowed formation of hetero-oligomeric complexes detected by immunoadsorption but the hGR complexes formed with mutated chsp90s were devoid of steroid binding properties. These results are consistent with (1) the possible involvement of the "A" region in the interaction of hsp90 with steroid receptors and (2) a role of B and Z regions in the hsp90 structure for maintaining the steroid binding property of the hGR.

Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.

Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.

Mutations in the "zinc fingers" or in the N-terminal region of the DNA binding domain of the human glucocorticosteroid receptor facilitate its salt-induced transformation, but do not modify hormone binding.

While the effects of the ligand (hormone) binding domain (LBD) on other receptor domain functions are known, the effects of other domains on LBD functions have not been studied. In this work, we examined the importance of the structural integrity of other domains of the human glucocorticosteroid receptor (hGR) on LBD activity (stability of 8S complexes, binding of hormone, and transformation from the 8S to the 4S form). Several mutations introduced outside the LBD affect neither the formation of stable 8S heterooligomeric complexes nor the hGR binding affinity for the agonist triamcinolone acetonide (TA) or the antagonist RU486. However, some of them led to an easier salt-induced transformation of the 8S-hGR into a 4S form. Deletion of the second zinc finger of the DNA binding domain (DBD) facilitated 8S dissociation whether the ligand was TA or RU486. Deletion of the first zinc finger facilitated dissociation only in the presence of RU486, while replacement of PRO 416 (in the N-terminal region of the DBD) by ARG destabilized the 8S form only in the presence of TA. Variations in the salt-sensitivity of the mutated 8S GR complexes as a function of the ligand suggest that the DBD may interact functionally (if not physically) with the LBD. This interaction (possibly mediated by hsp90) could be influenced by minor structural differences between agonist and antagonist-GR complexes.

Heat shock protein 90 as a critical factor in maintaining glucocorticosteroid receptor in a nonfunctional state.

Previous work demonstrated that the ligand binding domain (LBD) was required to determine the formation of the cytosolic, untransformed, inactive, 8 S, heterooligomeric form of the human glucocorticosteroid receptor (hGR) which includes the 90-kDa heat shock protein (hsp90) (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguère, V., Cadepond, F.M., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). Truncations of hGR deleting all or almost all of the LBD give GR derivatives in the non-hsp90-interacting 4 S form able to stimulate transcription in a hormone-independent manner. To identify the LBD subregion(s) involved in 8 S formation, we analyzed the sedimentation behavior of hGR mutants with various LBD internal deletions and/or truncations transiently expressed in cells that contain hsp90 but very low levels of endogenous GR, and we correlated the results with their transcriptional activity. LBD has been divided into three subregions: two of them, LBD1 (between amino acids 551 and 626) and LBD2 (between amino acids 627 and 696), include amino acid sequences highly conserved in the steroid receptor superfamily, and LBD3 consists of the carboxyl-terminal part of the molecule (amino acids 697-777). Each of these subregions can be deleted without impeding 8 S heterooligomer formation, and the corresponding receptors do not have transcriptional activity in the absence as well as in the presence of hormone. When linked to hGR mutants truncated after amino acids 532 or 550, each subregion does separately promote 8 S heterooligomeric complex formation and repress the intrinsic constitutive transcriptional activity of the truncated receptors. These 8 S complexes contain hsp90. In a control experiment, the linkage of 1,017 amino acids of beta-galactosidase to the carboxyl-terminal of 1-532 hGR gave a hybrid receptor still constitutively transcriptionally active which did not bind hsp90. These results provide evidence that there is a strong correlation between the association with hsp90 and the loss of GR functional properties and that hsp90 may play a critical role in maintaining the receptor in a nonfunctional state.